H.F. De Brabander

Recent developments in the use and abuse of growth promoters

During the last few years, control within the European Union (EU) for illegal growth promoters in cattle and pigs revealed only a limited number of positives. Analysis of illegal preparations, however, showed that steroids, often (esters of) natural hormones, and -agonists are still used. Corticosteroids, controlled to a much lesser extent, seem to have become the most important group, while even thyreostats remain. Alarming information was obtained from specific investigations in which a large variety of products were found, some of which had never been reported to be misused in the field of growth promotion. For -agonists and quinoxaline compounds, analogues of known compounds are synthesised. Other compounds are readily available as they are registered as growth promoters in some countries outside Europe or are allowed for specific veterinary purposes. Some classes of veterinary drugs are misused for their secondary pharmacological effects, e.g. benzodiazepines as feed intake enhancers and non-steroidal anti-inflammatory drugs (NSAIDs) as pale meat-making agents. Several non-traditional substances are suspected to be used in the field of breeding animals. This is the case for growth hormones (GHs) and all substances acting over this anabolic compound, as for instance, orally GH secretagogue. Moreover, ecdysteroids, which according to old Russian studies, have anabolic activity, are actually very easy to purchase on the Internet. Recent findings in different classes of growth promoters are discussed in detail.

Novel analytical methods for the determination of steroid hormones in edible matrices

This paper reviews recently published multi-residue chromatographic methods for the determination of steroid hormones in edible matrices. After a brief introduction on steroid hormones and their use in animal fattening, the most relevant EU legislation regarding the residue control of these substances is presented. An overview of multi-residue analytical methods, covering sample extraction and purification as well as chromatographic separation and different detection methods, being in use for the determination of steroid hormones (estrogens, gestagens and androgens), is provided to illustrate common trends and method variability. Emphasis was laid on edible matrices and more specifically on meat, liver, kidney, fat and milk. Additionally, the possibilities of novel analytical approaches are discussed. The review also covers specific attention on the determination of natural steroids. Finally, the analytical possibilities for phytosterols, naturally occurring steroid analogues of vegetable origin and a specific group of steroid hormones with a hemi-endogenous status are highlighted.

Residue analysis: future trends from a historical perspective.

A residue is a trace (microg kg(-1), ng kg(-1)) of a substance, present in a matrix. Residue analysis is a relatively young discipline and a very broad area, including banned (A) substances as well as registered veterinary medicinal products (B substances). The objective of this manuscript is to review future trends in the analysis of residues of veterinary drugs in meat producing animals out of historical perspectives. The analysis of residues in meat producing animals has known a tremendous evolution during the past 35-40 years. In the future, it can be foreseen that this evolution will proceed in the direction of the use of more and more sophisticated and expensive machines. These apparatus, and the necessary human resources for their use, will only be affordable for laboratories with sufficient financial resources and having guarantee for a sufficient throughput of samples.

Presence and metabolism of the anabolic steroid boldenone in various animal species : a review

The review summarizes current knowledge on the possible illegal use of the anabolic steroid boldenone. The presence of boldenone and metabolites in different animal species and the possibility of the occurrence of endogenous boldenone and metabolites is assessed, as are the methods of analysis used for detection. Different laboratories in the European Union have examined the occurrence of boldenone and its metabolites. The results were discussed at different meetings of a European Commission DG-SANCO Working Party and summarized in an expert report. The situation of the different laboratories at this time is also covered herein. The overall conclusion of the Working Party was that there was a necessity for further research to distinguish between naturally occurring and illegally used boldenone forms. The confirmation of the presence of boldenone metabolites (free and conjugated forms) in certain matrices of animals is proposed as a marker for the illegal treatment with boldenone.

Presence and metabolism of endogenous androgenic-anabolic steroid hormones in meat-producing animals: a review

The presence and metabolism of endogenous steroid hormones in meat-producing animals has been the subject of much research over the past 40 years. While significant data are available, no comprehensive review has yet been performed. Species considered in this review are bovine, porcine, ovine, equine, caprine and cervine, while steroid hormones include the androgenic–anabolic steroids testosterone, nandrolone and boldenone, as well as their precursors and metabolites. Information on endogenous steroid hormone concentrations is primarily useful in two ways: (1) in relation to pathological versus ‘normal’ physiology and (2) in relation to the detection of the illegal abuse of these hormones in residue surveillance programmes. Since the major focus of this review is on the detection of steroids abuse in animal production, the information gathered to date is used to guide future research. A major deficiency in much of the existing published literature is the lack of standardization and formal validation of experimental approach. Key articles are cited that highlight the huge variation in reported steroid concentrations that can result when samples are analysed by different laboratories under different conditions. These deficiencies are in most cases so fundamental that it is difficult to make reliable comparisons between data sets and hence it is currently impossible to recommend definitive detection strategies. Standardization of the experimental approach would need to involve common experimental protocols and collaboratively validated analytical methods. In particular, standardization would need to cover everything from the demographic of the animal population studied, the method of sample collection and storage (especially the need to sample live versus slaughter sampling since the two methods of surveillance have very different requirements, particularly temporally), sample preparation technique (including mode of extraction, hydrolysis and derivatization), the end-point analytical detection technique, validation protocols, and the statistical methods applied to the resulting data. Although efforts are already underway (at HFL and LABERCA) to produce more definitive data and promote communication among the scientific community on this issue, the convening of a formal European Union working party is recommended.

A validated analytical method for the determination of perfluorinated compounds in surface-, sea- and sewagewater using liquid chromatography coupled to time-of-flight mass spectrometry

Perfluorinated compounds (PFCs), which are extensively used in a wide variety of applications because of their specific surfactant properties, have recently appeared as an important new class of global environmental pollutants. Quantitative analysis of PFCs in aqueous matrices remains, however, a challenging task. During this study, a new analytical method for the determination of 14 PFCs in surface-, sewage- and seawater was developed and validated. The target analytes were extracted using solid-phase extraction followed by liquid chromatography coupled to a time-of-flight mass spectrometer (LC-ToF-MS). The use of very narrow mass tolerance windows (< 10 ppm) resulted in a highly selective MS-technique for the detection of PFCs in complex aqueous matrices. Validation of this analytical method in surface-, sewage- and seawater resulted in limits of quantification (LOQs) varying from 2 to 200 ng L⁻¹, satisfying recoveries (92-134%), and good linearity (R²=0.99 for most analytes). Analysis of samples of the North Sea, the Scheldt estuary, and three harbours of the Belgian coastal region led to the detection of four different PFCs. Perfluorooctane sulfonate (PFOS) was found to be the most abundant PFC in levels up to 38.9 ng L⁻¹.

Analytical possibilities for the detection of stanozolol and its metabolites

In sports doping, as well in man as in horseracing, stanozolol (Stan) was abused and became the subject of metabolism research. Also in veterinary practice, stanozolol became an important misused anabolic steroid. Like most other anabolic steroids, stanozolol has poor gas chromatographic behavior. It is difficult to detect in urine, because of low urinary excretion and renal clearance. This is due to the rapid metabolization, leading to low concentration levels of the parent compound found in urine. Therefore, most research studies have focused on the detection of its urinary metabolites. For the identification of the metabolites, different methods of extraction and detection are described in the literature. These are reviewed in this article. Most authors use a hydrolysis to free the phase II metabolites. Extraction procedures vary from solid-phase extraction (SPE), liquid-liquid (L-L) extraction to immunoaffinity chromatography (IAC). For the final detection, the use of gas chromatography (GC)-mass spectrometry (MS) can be compared with liquid chromatography (LC)-MSn. Different metabolites are identified depending on the administration of stanozolol in the animal experiment (oral or intramuscular). Analyses for these analytes in other matrices are also briefly discussed.

Development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method for quantifying thyreostats in urine without derivatisation

Thyreostatic drugs, illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981 (Council Directive 81/602/EC). For monitoring their illegal use, sensitive and specific analytical methods are required. In this study an UHPLC-MS/MS method was described for quantitative analysis of eight thyreostatic drugs in urine, this without a derivatisation step. The sample pretreatment involved a reduction step with dithiothreitol under denaturating conditions at 65 degrees C, followed by liquid-liquid extraction with ethyl acetate. This analytical procedure was subsequently validated according to the EU criteria (2002/657/EC Decision), resulting in decision limits and detection capabilities ranging between 1.1 and 5.5 microg L(-1) and 1.7 and 7.5 microg L(-1), respectively. The method obtained for all, xenobiotic thyreostats, a precision (relative standard deviation) lower than 15.5%, and the linearity ranged between 0.982 and 0.999. The performance characteristics fulfill not only the requirements of the EU regarding the provisional minimum required performance limit (100 microg L(-1)), but also the recommended concentration fixed at 10 microg L(-1) in urine set by the Community of Reference Laboratories. Future experiments applying this method should provide the answer to the alleged endogenous status of thiouracil.

Ion chromatographic determination of perchlorate in cattle urine

Abstract Much research has been done over the years on the subject of thyreostatic drugs of the thiouracil type in cattle fattening. These substances increase body weight by enlarged filling of the gastro-intestinal tract and by augmented water retention. On the other hand, little work has been done on the elimination and the presence of residues of inorganic thyreostatics such as perchlorate. The aim of this study was to evaluate the elimination of ammonium perchlorate in cattle urine according to the concentration given and the duration of administration. Perchlorate concentrations were measured by mobile phase ion chromatography. The detection limit was 0.1 mg kg −1 for tenfold diluted urine.

Detection of antithyroid residues in meat and some organs of slaughtered animals

A simple method is described for the routine detection of antithyroid residues in thyroid, liver, kidney and meat contaminated at levels as low as 10 ppb (10 parts per 10(9)). Tissue samples (2 g) are homogenized in methanol, contaminating lipids and amino acids are removed and the antithyroid residues are subjected to reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in buffer. The NBD derivatives are extracted with diethyl ether and separated by thin-layer chromatography. After spraying with cysteine or mercaptoethylamine, the antithyroid residues appear as fluorescent spots. The detection limit of these compounds is of the order of 200 pg.